by - OBJECTIVE:
To lay down a procedure for Operation & Calibration of HPLC.
- SCOPE:
This procedure is applicable for procedure for Operation & Calibration of HPLC.
- RESPONSIBILITY:
QC Officer/ QC Executive
- ACCOUNTABILITY:
QC Manager.
- PROCEDURE:
- Operation
- Check the general cleaning of the instrument.
- Switch on the instrument allow displaying the “MENU” screen.
- Keep the mobile phase bottles after filtration and degas.
- Purge the mobile phase line by pressing the “PURGE” button provided below to the display on the instrument.
- Connect the HPLC column whichever required, as per flow direction mentioned in the column.
- Set the flow rate. Column oven temperature and initial wavelength on system display.
- Operation
Calibration:
Once inSix month and after every maintenance job.
Materials and equipments for calibration:
- Caffeine
- Calibrated Stop Watch
- Calibrated thermometer
- 25 ml, 50 ml, 100, Class “A” volumetric flask.
- 1, 2, 5, 20 ml pipette Class “A”
- Column C18, 4.6 x 150 mm, 5 µm
- HPLC Grade Water
- HPLC Grade Methanol.
Calibration of Flow Rate
Take a 10 ml clean and dry Class “A” volumetric flask. Prime solvent port A with fully degassed Purified water. Set the vacuum degasser to run continuously. Prepare the calibrated stopwatch to begin timing. Enter 5.000 as the flow rate and run the pump. When the flow and pressure are stable, simultaneously insert the outlet tubing into the volumetric flask and start the stopwatch. stop the stopwatch when the bottom of the meniscus reaches the 10-mL mark on the flask. Record the elapsed time in seconds and the observed system pressure .
Calculate the flow rate using the following equation:
Volume of flask
Calculated flow rate = —————————————- x 60
Measured time in sec
Record the calculated flow rate.
Repeat the procedure for 1.0 ml/min, and 0.50 ml/min and record the observation.
Similarly repeat the procedure for other 3 port (port B, port C and Port D) also.
Record the calculated flow rate as per Format.
Acceptance criteria
Measured flow rate should be as under ± 2 % of set value:
| Set Flow Rate | Measured Flow Rate (ml/min.) |
| 5.0 ml/min | 4.90-5.10 |
| 1.0 ml/min | 0.98-1.02 |
| 0.5 ml/min | 0.49-0.51 |
Calibration of the Gradient Proportioning Valve (GPV)
Prepare a solution of Caffeine in Methanol: Dissolve about 25 mg of caffeine 100 ml of volumetric flask and dissolved in Methanol and further dilute 20 ml of this solution to 500ml with Methanol.
Fill the reservoir A and B with 100% Methanol.
Fill the reservoir for the C and D solution containing caffeine in Methanol.
Prime the pump using the solvent from all the lines individual for about 5 min with flow rate of 3.0ml.
Connect the union in place of column.
Create an instrument method using the following gradient program.
| Time | Module | Command | Value |
| 2:00 | Pumps | Solvent B | 50 |
| 2.00 | Pumps | Solvent C | 0 |
| 2.00 | Pumps | Solvent D | 0 |
| 2.01 | Pumps | Solvent B | 0 |
| 2.01 | Pumps | Solvent C | 50 |
| 2.01 | Pumps | Solvent D | 50 |
| 6.00 | Pumps | Solvent B | 0 |
| 6.00 | Pumps | Solvent C | 50 |
| 6.00 | Pumps | Solvent D | 50 |
| 6.01 | Pumps | Solvent B | 50 |
| 6.01 | Pumps | Solvent C | 0 |
| 6.01 | Pumps | Solvent D | 0 |
| 10.0 | Pumps | Solvent B | 50 |
| 10.0 | Pumps | Solvent C | 0 |
| 10.01 | Pumps | Solvent B | 45 |
| 10.01 | Pumps | Solvent C | 10 |
| 12.00 | Pumps | Solvent B | 45 |
| 12.00 | Pumps | Solvent C | 10 |
| 12.01 | Pumps | Solvent B | 50 |
| 12.01 | Pumps | Solvent C | 0 |
| 14.00 | Pumps | Solvent B | 50 |
| 14.00 | Pumps | Solvent C | 0 |
| 14.00 | Pumps | Solvent D | 0 |
| 14.01 | Pumps | Solvent B | 45 |
| 14.01 | Pumps | Solvent C | 0 |
| 14.01 | Pumps | Solvent D | 10 |
| 16.00 | Pumps | Solvent B | 45 |
| 16.00 | Pumps | Solvent D | 10 |
| 16.01 | Pumps | Solvent B | 50 |
| 16.01 | Pumps | Solvent D | 0 |
| 20.00 | Controller | Stop |
Set the detector to 254 nm.
Run single injections with 0.1 mL injection volume for 20 minutes with 2.0 ml flow rate .Click the prepare injection and then inject. Prepare the Processing method and save the processing method. Process the GPV run and create custom fields on 100% Height C/D, 10% of A/B/C and 10% of A/B/D. Check that the peak height is based on the average height along the flat area at the top of the peak.
Acceptance Criteria:
It should be 10% ± 1.0%.
GPV Calibration Calculation:
(Observed Height of C and D)
GPV value =———————————————- *100
Full scale Height A and B)
Injector Linearity
Use the following chromatographic conditions:
Column : C18, 250 mm x 4.6 mm, 5 µm
Flow Rate : 1.0 ml / min
Detection : UV 272 nm
Colum Temperature : Ambient
Sample Temperature : 25°C
Run Time : 10 min
Mobile phase:
Filtered and degassed mixture of 40 volumes of methanol and 60 volumes of water.
Test preparation:
Weigh and transfer about 50.0 mg of caffeine working standard to a 100 ml volumetric flask. Add 60 ml Mobile Phase, dissolve with the help of sonication and make up the volume with the same solvent (500 ppm). Dilute 2 ml of the resulting solution to 50 ml with Mobile Phase (20 ppm).
Make the system ready as given in procedure for operation.
Equilibrate the column, fill the vial with test solution, and put in sample tray.
Inject duplicates injection each of 5 µl, 10 µl, 20 µl, 50 µl and 100 µl of the test solution.
Calculate the coefficient of determination for different set of injection volumes.
Record the observation.
Acceptance criteria:
The Correlation Coefficient of determination should be greater than 0.999.
Volume Accuracy
- Use the following chromatographic conditions:
Column : C18, 250 mm x 4.6 mm, 5 µm
Flow Rate : 1.0 ml / min
Detection : UV 272 nm
Colum Temperature : 25°C
Sample Temperature : 25°C
Run Time : 10 min
Injection Volume : 50 µl
- To ensure the accuracy of this test, use forceps or wear lint-free gloves to handle the vial in this test.
- Fill a standard 1.5 ml vial with 1 ml of degassed HPLC-grade water.
- Seal the vial with a septum and cap.
- Zero the calibrated analytical balance, and then carefully weigh the vial (using forceps or gloved hand to move the vial). Record the weight (W1).
- Place the weighed vial in the sample rack and create the batch file.
- Fill reservoir A with degassed 100 % HPLC-grade water, and then prime the solvent delivery system for 20 to 30 min prior to start.
- Set the flow rate to 1.0 ml/min of degassed 100% HPLC-grade water.
- Create the batch sequence with 6 injections of 50µl from 1st vial, 2nd vial & 3rd vial one by one. Run the batch sequence from the menu and record the chromatograms.
- After completion of injections remove and reweigh the vial. Record the weight (W2). Use following formula to calculate the average volume of water injected per injection:
(W1- W2)
= ———————-x 1000 = µl/injection
6 x 0.99602
- Note: Water is used for this test because its density, 0.99823 g/ml at 20°C and 0.99602 g/ml at 25°C, introduces less than 0.3% error when volume is assumed equal to weight(grams x 1000 = µl)
- Record the results.
- Acceptance criteria:
- The measured value should be 50 µl ± 1.0 µl.
Injector Precision:
- Use the following chromatographic conditions:
Column : C18, 250 mm x 4.6 mm, 5 µm
Flow Rate : 1.0 ml / min
Detection : UV 272 nm
Colum Temperature : 25°C
Sample Temperature : 25°C
Run Time : 10 min
Inject 5 mL of 20 ppm Caffeine solution six replicates.
Acceptance criteria:
RSD of area counts of six replicates injections should be NMT 2.0 %.
Flow Rate Consistency:
RSD of retention time of six replicates injections under Injector Precision should be NMT 1.0%.
Auto Sampler Carryover:
Follow instrument condition as per above procedure.
Inject the 20µl volume of blank injection followed by caffeine solution (500ppm) and then inject blank injection.
Observe any peak eluting at the retention time of caffeine in last blank. Calculate the carry over as per the formula:
Area of peak in blank solution
= ——————————————————————— x 100
Area of peak due to caffeine in 500ppm caffeine solution
Record the observation.
Acceptance criteria:
The auto sampler carryover NMT 0.01 %.
Wavelength accuracy:
- Use the following chromatographic conditions:
Column : Column / Capillary
Flow Rate : 1.0 ml / min
Detection : UV 272 nm
Colum Temperature : 25°C
Sample Temperature : 25°C
Run Time : 10 min
Chromatographic Condition: Follow instrument condition as per except wavelength.
Prepare the instrument method at each wavelength from 201 nm to 209 nm, 241 nm to 249 nm and 269 nm to 277 nm.
Inject 20µl volume of 20 ppm solution (prepared by diluting the 2ml of 500ppm solution to 50ml in Mobile Phase) of Caffeine prepared in Mobile Phase by injecting single injection at each wavelength and record the response.
Record the observation in calibration report.
Acceptance criteria:
The maximum absorbance should be at 205 ± 2nm & 273 ± 2 nm. The Minima of peak response of caffeine should be obtained at 245± 2 nm.
Calibration of Column Heater Temperature
Place a calibrated digital thermometer inside the column heater chamber, near the top of the chamber. Ensure that the probe tip does not contact any surfaces. Close the chamber door.
Select the Column Temperature set field and enter 25°C and record the observed temperature . Similarly, enter 40°C and 75°C and record the observed temperature . Allow sufficient time for the chamber temperature to reach and stabilize at the set temperature.
Record the observed temperature.
Acceptance Criteria:
The observed value should be ± 2.0°C of set point temperature.
Detector Linearity
Use the following chromatographic conditions:
Column : C18, 250mm x 4.6 mm, 5 µm
Flow Rate : 1.0 ml/min
Detection : UV 272 nm
Colum Temperature : Ambient
Sample Temperature : 25°C
Injection volume : 20µl
Run Time : 10 min
Mobile phase:
Filtered and degassed mixture of 40 volumes of Methanol and 60 volumes of water. To perform the detector linearity test prepare 5 ppm, 10 ppm, 20 ppm, 50 ppm and 100 ppm concentration solutions of caffeine in Mobile Phase.
Preparation of Standard Solution (weight/dilution):
50 mg caffeine → 100ml (Mobile Phase) (500ppm)
Dilute 1 ml of 500ppm → 100ml (Mobile Phase) (5ppm)
Dilute 1 ml of 500ppm → 50ml (Mobile Phase) (10ppm)
Dilute 1 ml of 500ppm → 25ml (Mobile Phase) (20ppm)
Dilute 5 ml of 500ppm → 50ml (Mobile Phase) (50ppm)
Dilute 5 ml of 500ppm → 25ml (Mobile Phase) (100ppm)
Inject duplicates injection each of 5 ppm, 10 ppm, 20 ppm, 50 ppm and 100 ppm of the test solution.
Calculate the coefficient of determination for different set of injection volumes.
Record the observation.
Acceptance Criteria:
The Correlation Coefficient of determination should be greater than 0.999.
Noise and Drift Test:
Prepare Mobile Phase as blank and Inject 06 replicates.
Acceptance criteria: The noise should not be more than 300 µV
Drift should not be more than ± 1000 µV /h
6.7. Reporting
Record each use in the HPLC logbook and column logbook.
Record maintenance information in logbook.
Record calibration data in HPLC calibration format.
Report all problems in the operation of the HPLC immediately to the Head QC.
Update calibration sticker.
Cleaning and Shutdown of the system:
After the analysis part is over, purge the system and wash the analytical column with purified water.
After washing the column with water and preserve the column using appropriate solvent.
Shutdown the HPLC system by switching off the system.
Disconnect the column used from the HPLC system and put the former in its place.
- TRAINING:
Trainer : Manager – Quality Control
Trainees : Staff of the QC departments
- DISTRIBUTION:
Master Copy : QA Department
Controlled Copy : QC Department
Display Copy : QC Department
- REFERENCES:
In-House
- ABBREVIATIONS:
| Abbreviations | Extended Form |
| SOP | Standard Operating Procedure |
| NA | Not Applicable |
| QC | Quality Control |
| Sr. No. | Serial Number |
| QA | Quality Assurance |
| DEPT. | Department |
- REVISION HISTORY OF CHANGE:
| Sr. No. | Date | Revision Details | Revision No. |
| 01 | NEW SOP | 00 |
https://pharm85.wp9.hostingraja.info/column-chromatography/
https://pharm85.wp9.hostingraja.info/chromatography/
https://pharm85.wp9.hostingraja.info/liquid-chromatography/
https://pharm85.wp9.hostingraja.info/column-chromatography-principle/
